Method, target and application for NO accumulation to decrease Pseudomonas aeruginosa invasiveness

ABSTRACT

Provided are a method, target and application for NO accumulation to decrease invasiveness of Pseudomonas aeruginosa; achieving NO accumulation by means of external NO donors or disruption of internal NO metabolism in an environment where Pseudomonas aeruginosa is growing or being cultured, in order to decrease production by Pseudomonas aeruginosa of the invasion factor pyocyanin. Utilizing even micro-quantities of NO can significantly inhibit Pseudomonas aeruginosa PCN (PCN synthesis down 82% with 60 μM SNP treatment); by inhibiting NO reductase, an enzyme related to NO metabolism, PCN synthesis can be significantly inhibited (the PAO1-Δnor strain has 84% PCN reduction). Using NO donors or inhibiting enzymes involved in Pseudomonas aeruginosa NO metabolism as a means of combating bacterial infectious disease does not affect the body&#39;s normal microflora like traditional antibiotics.

CROSS REFERENCE OF RELATED APPLICATION

This is divisional application of a non-provisional application Ser. No.15/770,469 with a filing date of Apr. 23, 2018, which is a nationalphase national application of an international patent application numberPCT/CN2016/102973 with a filing date of Oct. 22, 2016, which claimed thepriority of application number 201510697439.9 with a filing date of Oct.23, 2015 in China. The contents of these specifications, including anyintervening amendments thereto, are incorporated herein by reference.

BACKGROUND OF THE PRESENT INVENTION Field of Invention

The present invention relates to biomedicine technology, and moreparticularly to a method of NO (Nitric Oxide) accumulation for reducingthe invasiveness and target point of Pseudomonas aeruginosa and itsapplication thereof.

Description of Related Arts

Pseudomonas aeruginosa (PA) is an important opportunistic pathogen, andis considered to be one of the three lethal bacteria of hospitalinfectious diseases. In the case of low human immunity, for example,people suffering from chronic diseases, patients with organtransplantation or patients with large-area burns and the like, it cancause different acute or chronic infection and even lead to death,especially to patients with sensitive immune system and patients withcystic fibrosis (CF); more than 80% of CF patients die from theseinfections. Pseudomonas aeruginosa has strong resistance to mostantibiotics (cephalosporins, penicillins, fluoroquinolones, andaminoglycoside antibiotics). At present in clinical, the antibioticsonly target to protein synthesis, cell walls, DNA replication, cellmembrane, folic acid coenzyme and etc., the limitations are due to drugresistance. The invasiveness of bacteria refers to its ability to infectand cause disease, and the invasiveness of bacteria depends on thevirulence factors produced by the bacteria and the mechanism of damage.The pathogenic bacteria destroy the normal physiological functions ofthe host cell by releasing a variety of toxin proteins and even lead tothe cell death. In recent years, research studies have shown that theinvasiveness of bacteria can be used as a target point to develop newantibiotic, that bacterial infection diseases can be treated byinhibiting the pathogenicity of bacteria. Since there is no effect onbacteria growth, bacterial drug resistance caused by traditionalantibiotics can be reduced or avoided.

Pyocyanin (PCN) is an important invasive factor of Pseudomonasaeruginosa. PCN is a blue phenazine compound that can penetrate througha biological membrane easily and is commonly found in the sputum of CFpatients infected by Pseudomonas aeruginosa. Recent animal modelexperiments showed that PCN is a key compound in the infection processof Pseudomonas aeruginosa, and its virulence can significantly reducethe number of cells in target tissues and organs, while PCN can alsoinhibit ciliary peristalsis of respiratory epithelial cells and increaseintracellular peroxide production. It has been reported that somecompounds can inhibit the synthesis of PCN of Pseudomonas aeruginosa tovarious degrees. For example, catechin (2˜4 mM) can reduce the synthesisof PCN by 50%; eugenol (50˜400 μm) can reduce the synthesis of PCN by56%; Yunnan Baiyao (2.5 g/l) can reduce the synthesis of PCN by 76.5%.However, many of these compounds have problems of high drug dosage andlow efficiency in inhibiting the synthesis of PCN, and many of them havestrong bactericidal properties themselves, which will not only increasethe drug resistance of the strain, but also affect the growth of thenormal flora in the human body and cause damage to human health.

Flagella is another important invasive factor of Pseudomonas aeruginosa.Flagella is a fibroinous adhesin that readily adheres to the intima.Adhesion is the first step for pathogens to contact and infect cells andis closely related to pathogenicity. The completion of the adhesionprocess is mainly caused by the action of adhesin. Adhesins are specialstructures and related proteins that exist on the surface of bacteria,which facilitate the bacteria to adhere to host cells. The flagella makePseudomonas aeruginosa have chemical tropism on the matrix, so that thecells can perform an unorganized movement on the surface of the liquidmedium with low viscosity, that is swimming motility, and another kindof exercise performed on the viscous semi-solid medium of the watersample, that is, swarming motility. Flagella-mediated movement helpsbacteria to increase nutrient availability, escape toxic substances,transfer to appropriate hosts, and find proper fixation sites, soflagella-mediated movement is another important invasive factor ofPseudomonas aeruginosa.

SUMMARY OF THE PRESENT INVENTION

An object of the present invention is to provide a method of NO (NitricOxide) accumulation for reducing the invasiveness and target point ofPseudomonas aeruginosa and its application thereof. From the perspectiveof “reducing the invasiveness of bacteria”, a new way to effectivelyinhibit Pseudomonas aeruginosa invasiveness factors is realized. Itsrelated drug targets can be applied to the preparation of drugs.

The present invention is implemented by the following technicalsolutions:

A method of reducing invasiveness of Pseudomonas aeruginosa, under aliving environment or a culture environment of Pseudomonas aeruginosa,achieving NO accumulation through exogenous NO donor or endogenous NOmetabolism blockage, thereby reducing the production of the invasivenessfactor of pyocyanin of Pseudomonas aeruginosa;

reducing the mediated motility of flagella and fimbriae of Pseudomonasaeruginosa simultaneously.

The exogenous NO donor comprises a compound capable of directly orindirectly supplying NO to Pseudomonas aeruginosa, the exogenous NOdonor is present in the living or culture environment of Pseudomonasaeruginosa.

The control of the endogenous NO metabolism blockage includesinhibition, blocking, or enhancement of NO metabolism-related enzymes.

The endogenous NO metabolism blockage refers to: cloning a plasmid orvector that is capable of recombining with NO metabolism-related enzymesand causing loss of function of NO metabolism-related enzymes intoPseudomonas aeruginosa; or employing inhibitors of NO metabolism-relatedenzymes their enzyme activity such that the activities of enzymes arereduce or lost to achieve NO accumulation in Pseudomonas aeruginosa; Oremploying genetic recombination or agonists to enhance the enzymes thatfavor the production of endogenous NO to achieve NO accumulation inPseudomonas aeruginosa.

A target that can promote the reduction of the invasiveness ofPseudomonas aeruginosa, the target includes intracellular gene locusassociated with NO metabolism in Pseudomonas aeruginosa and enzymesassociated with NO metabolism;

The gene locus associated with NO metabolism, the enzymes associatedwith NO metabolism are inhibited or activated to achieve NOaccumulation, thereby reducing the production of the invasiveness factorof pyocyanin of Pseudomonas aeruginosa and/or reducing the motility ofPseudomonas aeruginosa.

An application of achieving NO accumulation through exogenous NO donoror endogenous NO metabolism blockage to reduce the production of theinvasiveness factor of pyocyanin of Pseudomonas aeruginosa and/or reducethe motility of Pseudomonas aeruginosa under a living environment or aculture environment of Pseudomonas aeruginosa.

The application of NO donor compounds in the preparation of a medicamentfor reducing the invasiveness of Pseudomonas aeruginosa.

The use of a plasmid, vector or compound that blocks the NO metabolismof Pseudomonas aeruginosa in the preparation of a medicament forreducing the invasiveness of Pseudomonas aeruginosa.

The application includes:

The use of NO metabolism-blocking compounds in the form of antagonism,inhibition or blockage of NO metabolism in the preparation of amedicament for reducing the invasiveness of Pseudomonas aeruginosa.

The application in the preparation of a medicament for reducing theinvasiveness of Pseudomonas aeruginosa through a key enzyme molecule inthe NO metabolic pathway as a target or an enzyme, a carrier, or acompound of a recombination target.

The application of a medicament or a carrier which utilizes NOmetabolism related enzyme of Pseudomonas aeruginosa, including NOreductase, nitrate reductase, nitrite reductase and NO synthase, as atarget in the preparation of a medicament for reducing an invasivenessof Pseudomonas aeruginosa.

The medicament is a drug against pulmonary cystic fibrosis orPseudomonas aeruginosa septicemia.

Compared with the existing arts, the present invention has the followingadvantageous technical effects:

1) The invention provides a method, target and application for reducinginvasiveness of Pseudomonas aeruginosa by using NO accumulation. Byutilizing a trace amount of NO donor or an endogenous NO accumulation,the biological synthesis of invasiveness factor of Pseudomonasaeruginosa is effectively inhibited. That Pseudomonas aeruginosa PCN canbe realized only by using a trace amount of NO (60 μm SNP treatment, PCNsynthesis is reduced by 82%) and has characteristics of high efficiency.Meanwhile, NO accumulation can also influence the mobility mediated bythe flagellar and IV-type bacteria of Pseudomonas aeruginosa, not onlythe invasiveness is inhibited, but also the pathogenicity range isreduced, that other methods of killing Pseudomonas aeruginosa can befacilitated. Thus this provides a possible new method and a series ofdrug targets for treating Pseudomonas aeruginosa infection.

2) According to the present invention, if the key enzyme Nor of the NOdownstream metabolism is inhibited, the same inhibition effect oninvasiveness as to the exogenous NO donor is produced (compared a mutantstrain with no Nor to a wild strain, the PCN synthesis of the mutantstrain is reduced by 84% and the movement of the bacteria can beinhibited). Therefore, the NO metabolism-related enzyme represented byNor will be an effective target for inhibiting NO accumulation and thusreducing the invasiveness of Pseudomonas aeruginosa, and is an effectivetarget for medicament preparation.

3) The NO donor of the present invention, such as sodium nitroprusside(SNP), has been used for clinical trial since 1929, and it is atraditional powerful, rapid vasodilator by itself and so there is noproblem in safety. Furthermore, the present invention further confirmsthat SNP has no antagonistic effect on traditional antibiotics. Givenits availability to promote NO accumulation and thereby reduce theinvasiveness of Pseudomonas aeruginosa, the use of SNP has been expandedto allow it to be used in combination with antibiotics for thepreparation of Pseudomonas aeruginosa inhibitors.

4) The present invention seeks new antibacterial substances from theperspective of “antibacterial invasiveness”. NO, on the premise ofnon-inhibiting and non-sterilizing bacteria, can inhibit theinvasiveness of pathogenic bacteria, does not bring pressure to thebacteria itself, and can reduce or avoid the emergence of drugresistance. Therefore, the use of NO accumulation in the preparation ofmedicaments to inhibit bacterial infections can help to solve theclinical challenges of drug resistance of bacteria.

5) The present invention utilizes NO donors, or through inhibiting theNO metabolism related enzyme of Pseudomonas aeruginosa, as a way tofight bacterial infections, so it will not affect the normal microbialpopulation of the human body like traditional antibiotics, and the humannormal microbial population has a very important role in human health.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an influence of NO donor SNP on growth of PAO1 and synthesisof PCN; (wherein A is the effect of different concentrations of SNP onthe growth of PAO1; and B is the effect of different concentrations ofSNP on PCN production by PAO1; C is the trend of the effect of differentconcentrations of SNP on PCN production by PAO1)

FIG. 2 shows the effect of 60 μm SNP on the growth of clinical isolationof Pseudomonas aeruginosa and PCN synthesis (A refers to the effect of60 μm SNP on the growth of clinical isolation of Pseudomonas aeruginosa;B refers to the effect of 60 μm SNP on PCN synthesis of Pseudomonasaeruginosa);

FIG. 3 illustrates Nor gene knockout and PCR, and resistance detection(A refers to gene structure of Nor and gen structure of Nor insertedwith GM resistance; B is PCR validation of Δnor strain; C is theresistance verification of the Δnor strain);

FIG. 4 shows the effect of nitric oxide reductase Nor on the growth ofPAO1 and PA515 (A is the difference in the growth of PAO1 and its Δnormutant PAN1; B is the difference in growth of P515 and its Δnor mutantPAN515);

FIG. 5 shows the effect of nitric oxide reductase Nor on the PCNsynthesis of PAO1 and PA515 (A is the difference in the PCN synthesis ofPAO1 and its Δnor mutant PAN1; B is the difference in PCN synthesis ofP515 and its Δnor mutant PAN515; C is the colorimetric comparison ofextracted PCN);

FIG. 6 shows the effect of Nor on the swimming activity and the drivenproperty of PAO1, A: Test tube puncture swimming experiment result; B: aflat-plate swimming experiment result; C: flat-plate driven propertyexperiment result;

FIG. 7 shows the survival curves of mice infected with PAO1-Δnor andPAO1 at a bacteria concentration of OD600 nm=0.1 and inoculation volumeof 500 μl.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is described in further detail below withreference to specific embodiments, which is for illustration only and isnot intended to be limiting.

The present invention provides a method of reducing invasiveness ofPseudomonas aeruginosa, under a living environment or a cultureenvironment of Pseudomonas aeruginosa, achieving NO accumulation throughexogenous NO donor or endogenous NO metabolism control, thereby reducingthe production of the invasiveness factor of pyocyanin of Pseudomonasaeruginosa.

The exogenous NO donor comprises a compound capable of directly orindirectly supplying NO to Pseudomonas aeruginosa, the exogenous NOdonor is present in the living or culture environment of Pseudomonasaeruginosa. Specifically, the present invention utilizes a trace amountof NO donor to inhibit the biosynthesis of pyocyanin (PCN), which is oneof the Pseudomonas aeruginosa invasiveness factors, and can reduce theinvasiveness of bacteria without killing the bacteria.

The control of the endogenous NO metabolism includes inhibition,blockage, or enhancement of NO metabolism-related enzymes. Theendogenous NO metabolism blockage refers to: cloning a plasmid or vectorthat is capable of recombining with NO metabolism-related enzymes andcausing loss of function of NO metabolism-related enzymes intoPseudomonas aeruginosa; or employing inhibitors of NO metabolism-relatedenzymes such that the activities of enzymes are reduce or lost toachieve NO accumulation in Pseudomonas aeruginosa; Or employing geneticrecombination or agonists to enhance the enzymes that favor theproduction of endogenous NO to achieve NO accumulation in Pseudomonasaeruginosa.

In particular, NO reductase (Nor), which is related to the accumulationof endogenous NO, is used as the representative element. The effect ofthis target on the biosynthesis of PCN was studied by constructingmutants lacking Nor. The results showed that the addition of traceamount of NO donor or inhibition target nor can significantly reduce thebiosynthesis of pyocyanin of Pseudomonas aeruginosa.

The present invention further provides a target that can promote thereduction of the invasiveness of Pseudomonas aeruginosa, the targetincludes intracellular gene locus associated with NO metabolism andenzymes associated with NO metabolism in Pseudomonas aeruginosa;

The gene locus associated with NO metabolism, the enzymes associatedwith NO metabolism are inhibited or activated to achieve NOaccumulation, thereby reducing the production of the invasiveness factorof pyocyanin of Pseudomonas aeruginosa.

Based on the above description, the present invention provides anapplication of achieving NO accumulation through exogenous NO donor orendogenous NO metabolism blockage to reduce the production of theinvasiveness factor of pyocyanin of Pseudomonas aeruginosa under aliving environment or a culture environment.

Further explanations of the invention are given below:

1) Use of trace amount of NO donors to effectively inhibit thebiosynthesis of an invasiveness factor of Pseudomonas aeruginosa

The Pseudomonas aeruginosa is a conditional pathogen, and the presentinvention relates to its model strain PAO1 and clinically isolatedstrain of Pseudomonas aeruginosa.

The invasiveness factor is the secondary metabolite pyocyanin ofPseudomonas aeruginosa, pyocyanin belongs to the phenazine compound andits molecular formula C₁₃H₁₀N₂₀.

A trace amount of exogenous NO donor is used to inhibit the biosynthesisof pyocyanin of Pseudomonas aeruginosa. The NO donor includes sodiumnitroprusside (Na₂Fe(CN)₅NO), nitrosoglutathione GSNO (C₁₀H₁₆N₄O₇S), andetc.

2) Use of selective inhibition of NO downstream metabolic targets toeffectively inhibit the biosynthesis of invasiveness factor ofPseudomonas aeruginosa

The key enzyme for the downstream metabolism of NO in Pseudomonasaeruginosa—the NO-reductase, acts to convert intracellular NO to N₂O soas to avoid negative effect on cells which is caused by excessive NOaccumulation.

The selective inhibition is that the mutant strain lacking Nor isconstructed by the gene knockout method in the present invention.

Embodiment 1: Determining Significant Inhibition Effect of NO Donor SNPon the Biosynthesis of Pyocyanin of Pseudomonas aeruginosa ThroughFlat-Plate Culture

According to this embodiment, the strain used is the Pseudomonasaeruginosa model strain PAO1 (which is more common, and is availablefrom various collection centers). This bacteria are obligate aerobicbacteria and is culture under 37° C. in common bacteria culture medium(such as LB medium) for 20˜24 h.

In this study, the effect of NO on the synthesis of PCN in bacterialcells is investigated through adding multiple sets of flat plate samplesof SNPs at different concentrations and one blank flat plate. The finalconcentrations of SNPs in each group are 20, 40, 60, 80, and 100 μMrespectively. After PAO1 is cultured for 22 hours, the PCN produced bythe cells in the flat plate is extracted with chloroform, condensed, andthen developed with hydrochloric acid, and the level of PCN produced bythe cells is determined according to the light absorption value of thecoloring solution at 520 nm.

The results are shown in FIG. 1. The results showed that as theconcentration of SNP in the flat plate increased, the synthesis of PCNfrom Pseudomonas aeruginosa is reduced significantly, and the trend ofreduction of PCN within the concentration range of 20 μM˜60 μM issignificant. When the concentration is greater than 60 μM, the decreaseis flattened. It can be seen that 60 μM is the relatively idealconcentration (shown in C in FIG. 1).

Embodiment 2: Determining Significant Inhibition Effect of SNP on theBiosynthesis of Pseudomonas aeruginosa PCN without Imposing Pressure onthe Growth of Bacteria Through Shake Flask Cultivation

Implementation Basis:

1) Detection of bacterial growth: The growth of bacterial cells isdetermined by the light absorption value at 600 nm every 2 hours

2) PCN detection: The PCN produced by the bacterial cells in thebacteria solution is extracted with chloroform, concentrated, and thendeveloped with hydrochloric acid, then the level of PCN produced by thebacteria is determined according to the light absorption value at 520nm.

Specific Implementation:

According to this embodiment, the strain used is the Pseudomonasaeruginosa model strain PAO1; the clinical strain is isolated fromShaanxi Provincial People's Hospital and is isolated by the followingmethod: Selective medium such as MacConkey agar (MAC) is inoculated toclinical specimen with normal flora or specimen collected from theenvironment; plain or blood agar media is inoculated to clinicalspecimen without normal flora such as blood, cerebrospinal fluid,puncture fluid, and etc. The oxidase of the bacteria is positive, canoxidatively decompose glucose and xylose, produce acid without producinggas, but do not decompose lactose and sucrose. Liquefied gelatin candecompose urea, nitrate reduction to produce nitrite and nitrogen isproduced, indole negative without producing H₂S, arginine dihydrolasepositive by using citrate. Based on the above physiological andbiochemical characteristics, it is determined that the isolated strainis Pseudomonas aeruginosa.

In this study, shake flask culture is used to determine the effect ofSNP on the growth of PAO1 and PCN biosynthesis. Multiple sets of sampleswith different concentrations of SNPs and one blank sample are used tostudy the effect of NO on cell growth and PCN synthesis. The finalconcentrations of SNPs in each group are 20, 60, 100, 140, 160, and 200μM respectively. Samples are taken every 2 hours during shake flaskculture for cell growth detection and PCN detection. The results areshown in A and B in FIG. 1. When the concentration of NO donor SNP isless than 100 μM, it has relatively small effect on the growth of PAO1strain of Pseudomonas aeruginosa. From 20˜100 μM, the synthesis of PCNdecreases significantly with increasing concentrations of SNPs, and theratio of PCN reduction to cell growth reduction at 60 μM is the largest.This shows that the SNP at 60 μM has the most significant inhibitoryeffect on the synthesis of PCN while having a relatively small effect oncell growth.

Finally, the same experiments are performed on clinical isolations ofPseudomonas aeruginosa and similar results are obtained, thusdemonstrating that co-culture with NO donor is universally applicable tothe inhibition of PCN synthesis of Pseudomonas aeruginosa (see FIG. 2,where the effect on growth and inhibition of PCN synthesis are shown inA and B respectively).

Embodiment 3: Inhibition of the Target of NO Reductase (Nor) andConstruction of Mutant Strains Lacking Nor

Principle of implementation: Knocking out the Nor gene: First, constructa plasmid containing a Nor gene with inserted resistant fragment. Atthis point, Nor is unable to express because of the insertion ofresistant fragment. This recombinant plasmid is introduced into PAO1 bytriparental strain hybridization. Since the recombinant plasmid and theNor-gene on the genome contain the same homology arms, the in-vitroinactivated Nor gene is inserted into the plasmid by homologousrecombination to replace the Nor-gene on the genome so that the PAO1cannot express the Nor gene and thus cannot synthesize Nor, therebyaffecting the normal metabolism of NO and achieving NO accumulation.

Specific Implementation:

First construct the primers at the two ends of the Nor, then obtain theNor gene by PCR. Then, through double digestion, connect and constructthe recombinant plasmid with inserted Gm resistance Nor gene: nor-pexGm.

After the target fragment nor-pexGm is obtained, purify and digest withdifferent enzymes, then connected to the correspondingly digestedplasmid pEX18-Amp or pEX18-Tc, transform to E. coli and screen positiveclones with LB plates containing X-gal and AMP (50 μg/ml) or TET (15μg/ml). After the enzyme digestion test (as shown in FIG. 3B), thelacZ-GM fragment derived from pZ1918-LacZ is further cloned into the PCRfragment of the recombinant plasmid (as shown in FIG. 3A, partialfragment in norBC) and transformed into E. coli. Screen and verifylacZ-GM fragment insertion direction by using LB solid plates containingGM (15 μg/ml), and finally construct into a recombinant plasmid for geneknockout (as shown in FIG. 3A, wherein the fragment associated with theNor gene is: norBC-GM fragment).

After the recombinant plasmid is transformed into E. coli, homologousrecombination occurs between the inserted fragment on the plasmid(norBC-GM fragment) and the homologous fragment on the genome ofPseudomonas aeruginosa, and triparental strain hybridization method isrequired. This plasmid is brought into the target strain PAO1 by thehelper plasmid of the triparental strain hybrid.

The triparental strain hybridization process is as follows:

1. The donor strain (E. coli containing the recombinant plasmid, 15μl/ml of GM) and the mediated bacteria (E. coli containing the helperplasmid pRK2013, 50 μg/ml of Kan) are cultured under 37° C. in 25 ml LBmedium respectively, shake culture at 200 rpm for 14 hours, therecipient strain (Pseudomonas aeruginosa) is inoculated in 25 ml of LBmedium and cultured at 42° C. and 200 rpm with shaking for 14 hours.

2. Transfer the bacteria into 50 ml sterile large centrifuge tube,centrifuge at 8000 rpm for 3 minutes, discarding the supernatant andthen wash with 0.5 ml PBS for one time and transfer to 1.5 mlmicrocentrifuge tube. After weighing, resuspend the cells in PBS(concentration 500 μg/ml). Mix the above three bacterial suspensions ina new centrifuge tube in a ratio of 1:1:1 and gently blow the bacteriaevenly.

3. Take 0.1 ml of well-mixed bacterial suspension and spot on the centerof the LB solid plate. At the same time, spot 0.1 ml suspension ofwild-type Pseudomonas aeruginosa as a negative control. Plates areplaced in a 37° C. incubator to culture for 8-12 hours.

4. Resuspend the cells on the plate with 1 m PBS. After properlydiluted, 100 μl is applied to PIA solid medium containing GM (150μg/ml), place upside down position in a constant temperature incubatorat 37° C. for 24 hours. At the same time, wild type Pseudomonasaeruginosa is also applied to a plate as a negative control.

Through a certain chance of homologous recombination in the bacteria, amutant strain missing the entire Nor gene is obtained. Screen out thismutant strain by a series of screening methods such as PCR andresistance culture (as shown in C of FIG. 3).

Hybrid Strains Confirmation Experiments and Results:

A primary recombinant Pseudomonas aeruginosa can be grown on PIA platescontaining GM and positive clones are selected for scribing on LB platescontaining 10% sucrose. Secondary recombinant mutants are verified byTET sensitivity and PCR test. The results show that the PAO1/Δnor strainis successfully constructed and labeled as PAN1. Referring to FIG. 3C,wherein 1 is a plate with GM resistance and only PAN1 strain grows onit, and 2 is a plate without antibiotic and both PAO1 and PAN1 strainscan grow.

Similarly, in order to verify the universality, a clinical strainlacking the Nor gene is constructed simultaneously according to theabove method.

Embodiment 4: Cultivation of ΔNor Mutants and Wild Strains by ShakeFlask to Determine that the Selective Inhibition of Target Nor canSignificantly Inhibit the Biosynthesis of Pseudomonas aeruginosa PCN anddoes not Stress the Growth of Bacteria

Principle of Implementation: The role of NO reductase, Nor, is toconvert intracellular NO to N₂O so as to prevent excessive intracellularNO accumulation. Therefore, Δnor mutants will continue to accumulateendogenous NO and produce some biological effects. The present inventiondetermines the effect of endogenous NO on its invasiveness factor PCN bycomparing the growth and production of PCN in a Δnor mutant strain and awild-type Pseudomonas aeruginosa.

Specific implementation: Culture both the PAO1Δnor mutant strain and thewild-type strain at the same time. Samples are taken every 2 hours forgrowth and PCN analysis. The results show that the mutant lacking Norhas relatively small changes in growth (the growth is reduced by 10%)but the synthesis of PCN is significantly reduced (PCN reduced by 84%,test results are shown in FIG. 4) when compared to the wild-type strain.

In order to verify universality, the same experiment is performed on theΔnor mutant of a clinical strain PA515 and the wild strain, and similarresults are obtained. This shows that endogenous accumulation of NO hasa significant inhibitory effect on PCN synthesis while it has relativelysmall effect on the growth of Pseudomonas aeruginosa (FIG. 5).

Currently, studies have shown that the invasiveness of Pseudomonasaeruginosa can be reduced by inhibiting the synthesis of PCN. Forexample, Gee W. Lau et al. (Gee W. Lau et al. 2004) found that PCNproduced by Pseudomonas aeruginosa plays a key role in the process ofmice infection. In this study, three PCN synthetase mutants (ΔphzB1,ΔphzM, and ΔphzS) are constructed and the ability of these mutants toproduce pyocyanin are significantly reduced. The subsequent animalexperiments showed that the number of bacterial cells infected by thesemutant strains in the lungs of mice and the mortality rate of mice aresignificantly lower than those of wild strains. The results of thesestudies indicate that the invasiveness of Pseudomonas aeruginosa can bereduced by inhibiting the synthesis of PCN. In combination with theabove embodiment which determines that NO accumulation can significantlyreduce the synthesis of Pseudomonas aeruginosa PCN, the presentinvention shows that NO accumulation can effectively reduce Pseudomonasaeruginosa invasiveness.

Embodiment 5: Determine the Selective Inhibition of Target Nor canSignificantly Inhibit the Movement Ability of Pseudomonas aeruginosa PCNThrough Motility and Mobility Tests

Principle of implementation: Pseudomonas aeruginosa has a single-endsingle flagellum and several IV-type pilus at the end of the cell, bothof which are important organ of movement. Flagella-mediated movementhelps the bacteria to increase nutrient availability, escape toxicsubstances, transfer to appropriate hosts, and find suitable fixationsites. Flagella and type IV pili and their mediated movements areimportant pathogenic factors for Pseudomonas aeruginosa, both of whichalso play a crucial role in the initiation process of biofilms. Thepresent invention investigates the effect on the movement activity ofPseudomonas aeruginosa when selectively inhibiting target Nor throughmotility and mobility tests.

Specific Implementation:

Swimming motility test: Place a thin layer of swimming motilitydetection medium in a Petri dish [10 g/L tryptone, 5 g/L NaCl, 0.3%agarose, pH 7.0], and dry overnight at room temperature. Pick PAO1 andPAO1-Δnor cultured overnight on LB agar medium by using sterilizedtoothpick and inoculated onto the surface of the swimming detectionmedium. Cover the culture dish with a layer of plastic wrap and incubatefor 12-14 hours at 30° C. The bacteria relied on flagellar movement togrow around the inoculation site on the surface of the culture medium.The results show that the mutant strain lacking Nor significantlyreduces the length of movement and reduces the number of bacteriacapable of movement when compared with the wild strain, and the swimmingmotility is weakened. As shown in FIG. 6A, the comparison of WT and Δnoris illustrated.

Passive motility test: Place a thin layer of passive movement detectionmedium in a Petri dish [0.5% agarose, 8 g/L nutrient broth, 5 g/Lglucose, pH 7.0], and dry overnight. Pick PAO1 and PAO1-Δnor culturedovernight on LB agar medium by using sterilized toothpick and inoculatedonto the surface of the swimming detection medium. Cover the culturedish with a layer of plastic wrap and incubate for 12-14 hours at 30° C.Bacteria are affected by chemical tropism and relied on the movement offlagella and IV pili to form branched colonies on the surface of themedium. The results show that the mutant strain lacking Norsignificantly reduces the plaque diameter and the passive movementability when compared with the wild strain. As shown in FIGS. 6B and 6C,the comparison of WT and Δnor is illustrated.

Embodiment 6: Obtain Key Molecule and Key Site for NO Inhibition of PCNSynthesis by Using Quantitative Method of Phenyl NitrosylationModification

Principle of Implementation: The present invention utilizes theirreversible avidin labeling (IBP) method to affinity enrich the SNOmodified protein, analyzes the enriched SNO modified protein by massspectrometry, and combines literature data to obtain a possible SNOmodification key protein for NO inhibition on PCN synthesis.

Specific Implementation:

After processing Pseudomonas aeruginosa with NO donor treatment, collectthe bacteria and obtain protein of bacteria by cleavage; useirreversible biotinylation procedure (IBP) for blocking, reduction andbiotin labelling to produce Thionitrosylation-modified protein, which isthen enriched and modified with Streptavidin. Process elution ofmodified protein by 2.5% SDS. After separation by gel electrophoresis,enzymatic hydrolysis of protein gel into peptides. Labeled modifiedprotein peptides by mass spectrometric TMT reagents. Perform massspectrometry after desalting. Identify the protein that undergoes SNOmodification (nitrosylation) and quantitative changes after NO donorprocessing.

Protein modification studies show that: after treatment with NO donorSNP and GSNO (nitrosoglutathione), the overall SNO level of the PAO1protein in Pseudomonas aeruginosa strains is increased significantly.The results of mass spectrometry show that a number of proteins relatedto the synthesis of Pseudomonas aeruginosa pathogenic factors havedifferent degrees of SNO modification. The specific related proteins areshown in Table 6. The results show that NO inhibits the nitrosylationmodification process of multiple key proteins that regulate proteincontrol and protein synthesis, which is related to the synthesis of PCN,flagella and pilus.

TABLE 6 Ratio of target protein isotope 129/128 in relation to thePseudomonas aeruginosa pathogenic factor in the modification setsProtein name Protein category 129/128 Remark RhlR Regulation protein1.574 PCN, flagella and pili LasR Regulation protein 1.447 PCN, flagellaand pili PhzB Synthetic protein 1.388 PCN synthesis related PhzASynthetic protein 1.374 PCN synthesis related PilL Synthetic protein1.338 Pili synthesis related FimL Synthetic protein 1.352 Flagellasynthesis related

Note: 129 isotope labeled SNP treatment group with nitrosylated modifiedprotein samples, 128 isotope marker control group with nitrosylatedmodified protein samples, the value of 129/128 of the target protein inthe mass spectrometry test results represents: the relative change inthe thiosylated modification of this protein in the SNP treatment groupwhen compared with the control group.

Embodiment 7: Use of Animal Experiments to Study the In Vivo Effect ofNO on Pathogenicity of Pseudomonas aeruginosa

Principle of Implementation: The role of NO reductase, Nor, is toconvert intracellular NO to N₂O to prevent excessive intracellular NOaccumulation. Therefore, Δnor mutants will continue to accumulateendogenous NO and thus produce some biological effects. In the presentinvention, the effect of endogenous NO on the pathogenicity ofPseudomonas aeruginosa is determined by comparing the lethality of miceinfected with Δnor mutants strain and wild strain of Pseudomonasaeruginosa.

Specific Implementation:

Take about 100 ml of PAO1 and PAO1-Δnor original bacteria solution.Centrifuge at 3000 r/min and take the precipitation. Dilute to OD600 nm0.1 with sterile saline. Take 60 mice and randomly divide into threegroups based on body mass, which are: PAO1 group, PAO1-Δnor group, andblank control group. 0.5 ml per group, intraperitoneal injection ofbacterial suspension respectively. Observe and record the death of miceevery day for five days continuously. Use Log-Rank method to examine thedifferences between different groups. The results are shown in FIG. 7.The results show that the survival rate of the PAO1-Δnor group issignificantly higher than that of the PAO1 group (P=0.039<0.05 indicatessignificant difference between groups), and this shows that endogenousNO accumulation of in Pseudomonas aeruginosa can reduce thepathogenicity in the body.

Accordingly, the present invention provides the following applications:

The application of achieving NO accumulation through exogenous NO donoror endogenous NO metabolism blockage to reduce the production of theinvasiveness factor of pyocyanin of Pseudomonas aeruginosa and/or reducethe motility of Pseudomonas aeruginosa under a living environment or aculture environment of Pseudomonas aeruginosa.

The application of NO donor compounds in the preparation of a medicamentfor reducing the invasiveness of Pseudomonas aeruginosa.

The use of a plasmid, vector or compound that blocks the NO metabolismof Pseudomonas aeruginosa in the preparation of a medicament forreducing the invasiveness of Pseudomonas aeruginosa.

Since NO reductase is a method to achieve NO accumulation, other methodscan also achieve NO accumulation, its inhibition effect on PCN growth,its inhibition effect on Pseudomonas aeruginosa motility and itsreduction effect of Pseudomonas aeruginosa invasiveness should haveconsistent results.

In particular, the application includes:

The realization of NO metabolism-blocking compounds in the form ofantagonism, inhibition or blockade of NO metabolism in the preparationof a medicament for reducing the invasiveness of Pseudomonas aeruginosa.

The use of an enzyme, a carrier, or a compound that utilizes a keyenzyme molecule in the NO metabolic pathway as a target or a recombinanttarget in the preparation of a medicament for reducing the invasivenessof Pseudomonas aeruginosa.

The application of medicament or carrier that utilizes NO reductase ofPseudomonas aeruginosa as a target in the preparation of a medicamentfor reducing the invasiveness of Pseudomonas aeruginosa.

NO metabolism related enzyme of Pseudomonas aeruginosa also include NOreductase, nitrate reductase, nitrite reductase and NO synthase.Therefore, these enzymes (NO reductase, nitrate reductase, nitritereductase and NO synthase) can also be used as a target to control NOmetabolism, therefore also applicable in the preparation of a medicamentfor reducing an invasiveness of Pseudomonas aeruginosa.

Cystic fibrosis (CF) is a fatal autosomal recessive genetic disordercaused by a functional defect in the CFTR protein encoded by a mutationin the cystic fibrosis transmembrane regulator (CFTR) gene. There areabout 70,000 CF patients worldwide, of which the most common isCaucasian. Clinical data shows that the disease also has a certainincidence in our country. The mortality rate of CF is extremely high andthe median survival time is short (analysis of the clinical features ofChinese cystic fibrosis). It is urgent to find effective treatmentmeasures to improve the quality of life of CF patients. Spongysecretions from the lungs of CF patients block the airways and damagethe cilia scavenging system, causing repeated or persistent bronchialinfections. Pseudomonas aeruginosa is the most common colonizingbacteria in the airways. At the same time, Pseudomonas aeruginosainfection is also the most serious type of infection. According tostatistics, more than 90% of patients suffered from pulmonary cysticfibrosis die from chronic infection with Pseudomonas aeruginosa.

Pseudomonas aeruginosa septicemia is a clinically serious systemicinfection with serious illness and rapid progression. The mortality rateof Pseudomonas aeruginosa septicemia in foreign countries was 21.0% to39.0, which was higher than Staphylococcus aureus septicemia. Accordingto China's bacterial drug resistance monitoring network data from 2006to 2007, it shows that Pseudomonas aeruginosa accounted for 4.7% of thepathogens of bloodstream infections, and ranked third amongGram-negative bacteria (2006-2007 Mohnarin bloodstream infectionpathogen composition and drug resistance) Studying the prognosticfactors of Pseudomonas aeruginosa septicemia is essential for clinicalimprovement of treatment success rate.

Studies have shown that the top 5 isolates of pathogenic bacteriaobtained from CNS disease hospitals are: Studies have shown that the top5 isolates of pathogenic bacteria obtained from CNS disease hospitalsare: Staphylococcus aureus (23.7%), Pseudomonas aeruginosa (22.0%),Acinetobacter baumannii (18.6%), Klebsiella pneumoniae (8.5%),Pseudomonas maltophilia (6.8%). The foreign research statistics showthat the clinical manifestations of central nervous system infectioncaused by Pseudomonas aeruginosa have a poor prognosis and the mortalityrate is above 60%.

Therefore, based on an NO donor compound, the function of a medicamentor carrier which utilizes NO reductase of Pseudomonas aeruginosa as atarget to act on the invasiveness of Pseudomonas aeruginosa (reduce theproduction of the invasiveness factor of pyocyanin of Pseudomonasaeruginosa and/or reduce the motility of Pseudomonas aeruginosa) canalso be applied to the preparation of medicament against pulmonarycystic fibrosis or Pseudomonas aeruginosa septicemia.

The above-presented embodiments are preferred examples for implementingthe present invention, and the present invention is not limited to theabove embodiments. Any non-essential additions and replacements made bythose skilled in the art according to the technical features of thetechnical solutions of the present invention all belong to theprotection scope of the present invention.

What is claimed is:
 1. A method of achieving NO accumulation to reduce an invasiveness of Pseudomonas aeruginosa during an initial invasion stage, comprising the steps of: through exogenous NO donor or endogenous NO metabolism blockage, (a) reducing production of an invasiveness factor of pyocyanin of Pseudomonas aeruginosa, (b) reducing a motility of Pseudomonas aeruginosa, or (c) reducing production of an invasiveness factor of pyocyanin of Pseudomonas aeruginosa and reducing a motility of Pseudomonas aeruginosa under a living environment or a culture environment of Pseudomonas aeruginosa.
 2. The method according to claim 1, wherein the method is used to prepare a medicament for reducing an invasiveness of Pseudomonas aeruginosa by using the plasmid, the vector or the compound that blocks the NO metabolism of Pseudomonas aeruginosa during an initial invasion stage.
 3. The method according to claim 2, characterized in that: said method comprises the steps of: using NO metabolism-blocking compounds in the form of antagonism, inhibition or blockade of NO metabolism when preparing the medicament for reducing the invasiveness of Pseudomonas aeruginosa; using an enzyme, a carrier, or a compound that utilizes a key enzyme molecule in the NO metabolic pathway as a target or a recombinant target when preparing the medicament for reducing the invasiveness of Pseudomonas aeruginosa.
 4. The method according to claim 3, characterized in that: the medicament is a drug against pulmonary cystic fibrosis or Pseudomonas aeruginosa septicemia and Pseudomonas aeruginosa-induced central nervous system infection. 